Deck of CArGs.

The instructions for generating and sustaining all life forms are encoded within each life form’s genome. A major challenge in the postgenomic sequencing era has been assigning functions to the hundreds of thousands of noncoding sequence elements within our nuclear genome that are homologous to sequences in other species. These snippets of DNA have been hypothesized to impart information for initiating DNA replication, for interand intrachromosomal recombination, for structural integration of the genome into the surrounding nucleoskeleton, and for transcriptional regulation of gene expression. The latter regulatory elements have been of particular interest inasmuch as variations among our own species, and to some extent between species, are thought largely to be a function of differences in the timing, duration, and intensity of gene expression. Moreover, the explosive rise in single nucleotide polymorphism (SNP) association studies has generated a mounting number of noncoding SNPs whose functions, while poorly defined at this time, will undoubtedly include the regulation of gene expression.1 Finally, the continued discovery of regulatory elements controlling gene expression will augment our “genomic tool box” of reagents for expressing and inactivating genes in a context-dependent manner. Elucidating the function of all regulatory elements in our genome is therefore a critically important endeavor from both a clinical and basic science perspective. The 3 muscle types display unique patterns of gene expression; however, during development and in some pathological states there is overlap in gene expression profiles suggesting a common mode of regulation (Figure 1). For example, the majority of cyto-contractile genes expressed in each muscle cell type are under direct control of the widely expressed transcription factor, serum response factor (SRF). SRF self-dimerizes and binds to a 10-bp sequence known as a CArG element or CArG box (Figure 2). CArG boxes are found in the 5 promoter and intronic region of a rising number of cyto-contractile genes. Based on 20 years of DNA-protein and promoter analyses, as well as comparative genomics, we now recognize that SRF may potentially bind to 1216 permutations of a CArG box, with CCTTATATGG emerging as a consensus sequence (Figure 2). Recent genome-wide studies have further advanced our understanding of the base sequence character of CArG elements and have greatly expanded the so-called CArGome.2–5 As of this writing, more than 200 CArG boxes controlling expression of some 170 mammalian SRF target genes have been identified with more than 300 hypothetical CArG boxes awaiting wet-laboratory validation. SRF possesses relatively weak transcriptional activity, but binds to any one of 56 cofactors that potently activate target gene expression, mainly through alterations in chromatin permissive for DNA transcription. Many SRF cofactors exhibit cell-restricted patterns of gene expression during development and postnatal life. One of the more powerful cell-restricted SRF cofactors is myocardin (Myocd), which was first cloned in a bioinformatic screen for cardiac-specific genes.6 Expression of Myocd is highly specific for cardiac and SMCs, with transient expression in developing skeletal muscle precursors.6,7 Myocd forms a ternary complex with SRF-bound CArG boxes and, through its association with a variety of other coregulators of gene expression, directs expression of cardiac and SMC cyto-contractile genes.8–10 Though cardiac genes are induced when Myocd is ectopically expressed in nonmuscle cells, little evidence of a structural or functional cardiac muscle phenotype is manifest. In contrast, Myocd orchestrates structural, biochemical, and physiological characteristics of SMCs.11,12 Thus, Myocd appears to be the SMC equivalent of MyoD, the original master regulator of the skeletal muscle phenotype. SMCs are defined by a molecular signature of gene expression that includes genes encoding contractile, cytoskeletal, ion channel, transcription factor, and signaling proteins, all of which are essential to carry out the unique function of this cell type.13 The regulatory regions of many of these genes have been characterized both in vitro and in vivo and more than half contain functional CArG elements.14 Now, in this issue of Circulation Research, Petit et al report the discovery of an alternative form of the SMC-specific gene, LIM domain containing preferred translocation partner in lipoma (aka Lpp), that appears to be under direct control of CArG-SRFMyocd ternary complexes.15 Lpp protein expression was shown previously to be highly specific for SMCs where, in association with vinculin at peripheral dense bodies, it mediates cell migration.16 Petit et al sought to define whether any functional CArG boxes reside in or around the 588-kb mouse Lpp locus using a bioinformatics approach based on CArG nucleotide frequencies (Figure 2). A total of 35 CArG elements were found over the interrogated sequence, a number surprisingly lower than the theoretical frequency of 1 every 910 base pairs of DNA sequence. Three of the 35 CArG elements were found to be homologous with corresponding CArGs in several other species, though their position is more than 50 kb away from the annotated start site of Lpp transcription. Because virtually all functional CArG elements reside within a 4-kb window of transcription start sites,5 Petit The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Aab Cardiovascular Research Institute, University of Rochester School of Medicine & Dentistry, Rochester, NY. Correspondence to Aab Cardiovascular Research Institute, University of Rochester School of Medicine & Dentistry, 211 Bailey Road, West Henrietta, NY 14586. E-mail [email protected] (Circ Res. 2008;103:13-15.) © 2008 American Heart Association, Inc.